Epidemiological studies indicate that increased plasma cholesterol levels increase the risk for atherosclerosis. Five completed major trials have provided conclusive evidence of a benefit from treatment aimed primarily at reducing low-density lipoprotein (LDL)-cholesterol (Illingworth R D., et al. Current Opini. Lipidol. 1999, 10:383-386). Among other lipoprotein risk factors is familial dysbetalipoproteinemia, which results in the accumulation of remnant atherogenic lipoproteins derived from the catabolism of chylomicron and VLDL (Kwiterovich, P. O., Jr. Am. J. Cardiol. 1998, 82:3U-7U). It has been shown that a 1% decrease in the plasma cholesterol level decreases the risk of coronary artery disease by 2% (Deedwania, P. C. Med. Clin. North Am. 1995, 79:973-998). The focus of angiographic trials has been on LDL reduction and these studies have demonstrated that decreases in LDL-cholesterol of more than 30% to 35% are associated with lower rates of coronary events (Watts, G. W., et al. Atherosclerosis 1998, 414:17-30). There is also growing evidence that triglyceride-rich lipoproteins may adversely affect endothelial function and increase oxidative stress by promoting the production of small, dense LDL and by reducing high-density lipoprotein (HDL) levels (Marais, D., Curr. Opin. Lipidol. 2000, 11:597-602).
Apolipoprotein E (apo E) plays an important role in the metabolism of triglyceride-rich lipoproteins, such as very low density lipoprotein (VLDL) and chylomicrons. Apolipoprotein E mediates the high affinity binding of apo E-containing lipoproteins to the low density lipoprotein (LDL) receptor (apo B, E receptor) and the members of its gene family, including LDL receptor related protein (LRP), very low density lipoprotein receptor (VLDLR) and the apoE2 receptor (apoE2R) (Mahley, R. W., (1988) Science 240, 622-630). The putative and complex role of apo E in atherosclerosis has been emphasized by several observations: (i) mice that overexpress human apo E have lower levels of total plasma cholesterol levels (Shimono, H. N., et al., (1992) Eur. J. Clin. Invest. 90, 2084-2991), (ii) intravenous injection of human apo E into cholesterol-fed rabbits protects these animals from atherosclerosis (Yamada, et al., (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 665-669), and (iii) loss of the apo E gene in mice produces spontaneous atherosclerosis (Zhang, S. H., et al., (1992) Science 258, 468-471) which is ameliorated when macrophage-specific apo E expression is initiated in apo E-deficient mice (Spangenberg, J., et al., (1997) Biochem. Biophys. Acta 1349, 109-121).
Apolipoprotein E is a protein that binds lipid and has two major domains (Mahley, R. W., et al. J. Lipid Res. 1999, 40:622-630). The 22 kDa amino terminal domain has been shown by X-ray crystallographic studies to be a 4-helix bundle (Wilson, C., et al. Science 1991; 252:1817-1822) and to contain a positively-charged receptor binding domain. For this region to mediate very low-density lipoprotein (VLDL) binding to its receptors, the apolipoprotein must associate with the lipoprotein surface; this is enabled by the C-terminal amphipathic helical region. If the 4-helix bundle that contains the positively charged receptor-binding domain does not open up on the lipoprotein surface, then the VLDL is defective in binding to receptors. Thus, the positively charged arginine (Arg)-rich cluster domain of the Apo E and the C-terminal amphipathic helical domain, are both required for the enhanced uptake of atherogenic Apo E-containing lipoproteins.
Apo E is secreted as a 299 amino acid residue protein with a molecular weight of 34,200. Based on thrombin cleavage of apo E into two fragments, a two-domain hypothesis was initially suggested to explain the fact that the C-terminal region of apo E (192-299) is essential for its binding to hypertriglyceridemic VLDL and the N-terminal 22 kDa domain (1-191), binds to the LDL-R (Bradley, W. A., et al., (1986) J. Lipid Res. 27, 40-48). Additional physical-chemical characterization of the protein and its mutants have extended this concept and have shown that the region 192-211 binds to phospholipid while the amino terminal domain (1-191) is a globular structure that contains the LDL receptor binding domain in the 4-helix bundle (Wilson, C., et al., (1991) Science 252, 1817-1822). Studies with synthetic peptides (Sparrow et al.) and monoclonal antibodies pinpointed the LDL receptor binding domain of apo E between residues 129-169, a domain enriched in positively charged amino acids, Arg and Lys (Rall, S. C., Jr., et al., (1982) PNAS USA 79, 4696-4700; Lalazar, A., et al., (1988) J. Biol. Chem. 263, 3542-2545; Dyer, C. A., et al., (1991) J. Biol. Chem. 296, 22803-22806; and Dyer, C. A., et al., (1991) J. Biol. Chem. 266, 15009-15015).
Further studies with synthetic peptides were used to characterize the structural features of the domain of apo E that mediates its interaction with the LDL receptor (Dyer, C. A., et al., (1991) J. Biol. Chem. 296, 22803-22806; Dyer, C. A., et al., (1991) J. Biol. Chem. 266, 15009-15015; and Dyer, C. A., et al., (1995) J. Lipid Res. 36, 80-8). Residues 15 . 141-155 of apo E, although containing the positively charged residues, did not compete for binding of LDL in a human skin fibroblast assay, but did so only as tandem covalent repeats [i.e. (141-155)2]. N-acetylation of the (141-155)2 peptide, on the other hand, enhanced LDL binding to fibroblasts (Nicoulin, I. R., et al., (1998) J. Clin Invest. 101, 223-234). The N-acetylated (141-155)2 analog selectively associated with cholesterol-rich lipoproteins and mediated their acute clearance in vivo (Nicoulin, I. R., et al., (1998) J. Clin Invest. 101, 223-234). Furthermore, these studies indicated that the prerequisite for receptor binding is that the peptides be helical (Dyer, C. A., et al., (1995) J. Lipid Res. 36, 80-88). Enhanced LDL uptake and degradation were also observed (Mims, M. P., et al., (1994) J. Biol. Chem. 269, 20539-20647) using synthetic peptides modified to increase lipid association by N,N-distearyl derivation of glycine at the N-terminus of the native 129-169 sequence of Apo E (Mims, M. P., et al., (1994) J. Biol. Chem. 269, 20539-20647). Although LDL binding is mediated by the cationic sequence 141-155 of human Apo E, Braddock et al. (Braddock. D. T., et al., (1996) Biochemistry 35, 13975-13984) have shown that model peptides of the highly conserved anionic domain (41-60 of human Apo E) also modulate the binding and internalization of LDL to cell surface receptors. However, these peptides do not enhance LDL degradation.
Chylomicron is a lipoprotein found in blood plasma, which carries lipids from the intestines into other body tissues and is made up of a drop of triacylglycerols surrounded by a protein-phospholipid coating. Chylomicron remnants are taken up by the liver (Havel, R. J., 1985, Arteriosclerosis. 5:569-580) after sequestration in the space of Disse, which is enhanced in the presence of Apo E (Kwiterovich, P. O., Jr., 1998; Deedwania, P. C., 1995; and Watts, G. W., et al., 1998). Apo E is the major mediator of hepatic remnant lipoprotein uptake by the LDL receptor or LRP. Lipolysis of normal VLDL Sf (subfraction) of more than 60 permit binding of the lipolytic remnant to the LDL receptor (Catapano, A. L. et al., 1979, J. Biol. Chem. 254:1007-1009; Schonfield, G., et al. 1979. J. Clin. Invest. 64:1288-1297). Lipoprotein lipase (LpL) may facilitate uptake through localization of Apo B-containing lipoproteins to membrane heparan sulphate proteoglycan (HSPG) (Eisenberg, et al. 1992. J. Clin. Invest. 90:2013-2021; Hussain, M., et al., J. Biol. Chem. 2000, 275:29324-29330) and/or through binding to the LDL-receptor-related protein (LRP) (Beisiegel, U., et al., 1989, Nature 341:162-164). Cell-surface HSPG may also function as a receptor and has variable binding affinities for specific isoforms of Apo E. In particular, Apo E is synthesized by the liver and also by monocyte/macrophages, where it exerts its effect on cholesterol homeostasis. In vivo evidence for the local effect of lack of Apo E comes from the observations of Linton and Fazio, who showed accelerated atherosclerosis in C57BL/6 mice transplanted with bone marrow from Apo E-deficient mice (Linton, M. F. and Fazio, S. Curr. Openi. Lipidol. 1999, 10:97-105). Apo E-dependent LDL cholesteryl ester uptake pathway has been demonstrated in murine adrenocortical cells (Swarnakar, S., et al. J. Biol. Chem. 2001, 276:21121-21126). This appears to involve chondroitin sulphate proteoglycan (CSPG) and a 2-macroglobulin receptor.
It has been shown that the receptor-binding domain of Apo E, rich in Arg residues (141-150), covalently linked to a synthetic class A amphipathic-helical domain, enhances the hepatic atherogenic lipoprotein uptake (Datta, G., et al. Biochemistry 2000, 30:213-220). Recent studies indicate that a potential anti-atherogenic action of Apo E is that it stimulates endothelial production of heparan sulfate (HS) (Paka, L., et al. J. Biol. Chem. 1999, 274:4816-4823). Lipoproteins are complexes of one or more lipids bound to one or more proteins and transport water-insoluble fats in the blood. Cholesterol is carried through the bloodstream by lipoproteins. There are no agents available which reduce cholesterol via the binding mechanisms of lipoproteins. There is a need for more effective agents that are capable of reducing cholesterol in a subject so as to reduce diseases and conditions which are associated with increased cholesterol.
U.S. Pat. No. 6,506,880 denotes the first effort to synthesize apolipoprotein E-mimicking peptides based on the hypothesis that since lipid binding is essential for surface localization of the peptide on lipoproteins and for the receptor binding domain of apo E to be appropriately accessible to bind to the LDL receptor, joining a well-characterized, lipid-associating peptide such as the model class A amphipathic helix, 18A, to the 141-150 peptide sequence of apo E should be sufficient to confer biological activity. It was found that the peptides enhanced LDL/VLDL binding to a cell, increased LDL/VLDL degradation by a cell, lowered LDL/VLDL cholesterol in an in-need individual with atherosclerosis.
The present invention provides novel synthetic apolipoprotein E (ApoE)-mimicking peptides wherein the receptor binding domain of apolipoprotein E is covalently linked to 18A, the well characterized lipid-associating model class A amphipathic helical peptide as well as possible applications of the synthetic peptides in lowering human plasma LDL/VLDL cholesterol levels, thus inhibiting atherosclerosis. The present invention also provides possible applications of the synthetic peptides to improve HDL function and/or exert anti-inflammatory properties.